The separation device for separating the material to be separated from the mobile phase by passing the mobile phase containing the material to be separated through the stationary phase is used, for example, when separating useful materials produced by cell culture. For example, antibody drugs, which are the useful materials, can be obtained by culturing animal cells having antibody producibility and by separating and purifying antibodies secreted into a culture solution. That is, the useful materials such as antibody drugs are separated and purified using chromatography after removing cells from the culture solution. Meanwhile, when producing a given useful material by using microorganisms, the useful material often accumulates in the cells. In this case, solids are removed from the solution after the cells are disrupted, and then the useful material is separated and purified using chromatography.
The antibody drugs are generally produced through a clarification step, a capture step, an intermediate purification step, and a polishing step. In this production method, different separation devices, i.e., chromatographic methods are used in each step depending on the type of the antibody of interest. However, it is common that purity of the antibody is increased and selectivity of the antibody of interest is increased in a stepwise manner.
The clarification step is to remove the solids or proteins other than the antibody from the culture solution as much as possible. Since culture solution components such as serum, ascites fluid, and hybridoma cell culture solution are different and inclusions are also different for each type of the antibodies, the clarification is performed by using different methods such as salting-out, filtration with a filter, centrifugal separation.
Further, in the capture step, affinity chromatography is usually used. When the antibody of interest is IgG, very high specific affinity chromatography using Protein A or Protein G as a ligand is used, and it is possible to perform purification to a purity of 90% or more in one step. On the other hand, when the antibody of interest is, for example, IgM or IgY, which is an antibody having low affinity for Protein A and Protein G, affinity chromatography utilizing thiophilic interaction is used. Further, when the antibody of interest is IgA, IgD or IgE, affinity chromatography with a column immobilized with a secondary antibody recognizing the antibody is used, because blood concentration thereof is low and there is no high affinity ligand therefor. In the capture step, processing speed and processing capacity are important, and it is required to rapidly separate and concentrate the antibody of interest from a crude state of a cell extract or the like. It is for facilitating subsequent steps.
Next, in the intermediate purification step, contaminants recovered along with the antibody of interest in the capture step are removed. In this step, since an amount of treatment solution is large, ion exchange chromatography with large capacity is generally used. Note that, when the amount of treatment solution is small, the capture step and the intermediate purification step can be performed in one step.
The last polishing step is a step for separating the contaminants remaining slightly by using high performance column, to obtain final purified antibody. In the polishing step for obtaining the antibody of interest, gel permeation chromatography with a high resolution column is generally used. By using gel permeation chromatography, it is possible to perform buffer exchange together with removal of low-molecular materials inhibiting structural analysis.
In biopharmaceutical production, it is typical that a plurality of culture tanks with a volume of about 10 m3 are installed, and after culturing animal cells in the culture tanks, a large amount of culture solution (10 m3 or more per one batch) containing biopharmaceuticals (for example, antibodies) is processed. As a particular problem in a process of such a large amount of culture solution, the capture step using affinity chromatography can be mentioned. A protein (for example, Protein A), which is used in the affinity chromatography and is specifically bound to biopharmaceuticals, is very expensive. Therefore, the column with the protein is reused repeatedly for each culture batch. However, reuse of the column has a possibility that various components in the previous process remain, as well as a possibility of column degradation. Due to these possibilities, there is a possibility that purification quality of biopharmaceuticals varies among the culture batches.
In general, an affinity purification column for producing biopharmaceuticals is very large with a diameter of about 1 m and a bed height of several tens cm, and an equipment with the affinity column requires a certain space. Meanwhile, since the culture solution to be treated has a large volume of 10 m3 or more, it is necessary to repeat the capture step many times to process a batch of culture solution even when using the above large purification column. The capture step includes steps of (I) column equilibration, (II) target material adsorption, (III) washing, (IV) target material elution, and (V) column regeneration. A step of processing the culture solution is the step of (II) target material adsorption, and the steps of (I) and (III) to (V) are in a wait state for processing the culture solution. In order to process a large volume of culture solution, the steps of (I) to (V) must be repeated many times, and the wait state is long, and as a result, processing time in the capture step is increased. For example, in Patent Document 1, in order to eliminate waiting time, a method in which plural columns are installed, and the step of (II) target material adsorption for processing the culture solution is continuously performed by shifting the capture step in each column has been studied. However, in this method, it is necessary to ensure a large equipment space to install the plural columns, and there is a problem that cost of column filler is increased.